The area of microRNA biology is relatively new in our laboratory. However, we are interested in expanding the scope of our research to include microRNAs, since they are major regulators of gene expression but their influence during the stress and proliferative responses is poorly understood. We are particularly well poised to investigate microRNAs because their effects on mRNA stability and translation are similar in many respects to those of RBPs; therefore, we already have the cell systems, methodologies, and expertise in place to study them. To investigate microRNA levels and function in mammalian cells, we employ approaches such as microRNA overexpression (by transfecting microRNA precursors), microRNA reduction (by transfecting antisense microRNAs), and the identification of microRNA-associated mRNAs using biotinylated microRNAs to affinity-purify bound transcripts. We investigate whether microRNAs affect the stability of target mRNAs by measuring the steady-state levels and half-lives of the mRNAs of interest as a function of microRNA abundance. We investigate whether microRNAs affect the translation of target mRNAs by studying the relative assocation of the mRNA with translating polysomes and by quantifying the nascent translation rates of the encoded proteins. We also employ reporter constructs to gain additional insight into these processes.[unreadable] [unreadable] During this funding period, we have reported that miR-24 reduces the translation of p16 by associating at least with two segments of the p16 mRNA, one in its coding region, one in the 3-untranslated region, a tumor suppressor that blocks cell cycle progression by inhibiting cdk4/6. In the course of this investigation, we also identified other microRNAs whose levels were upregulated or downregulated during replicative senescence.[unreadable] [unreadable] Ongoing work is analyzing the mRNA targets of various senescence-associated microRNAs. Among the targets under study is MKK4, whose translation appears to be modulated jointly by several microRNAs. In addition, we are pursuing the analysis of microRNAs that potently modulates the translation of the RBP HuR. Other studies are focused on the influence of RBPs upon microRNA action and vice versa, using the c-Myc mRNA as the target transcript.